Research priorities to fill knowledge gaps on ASF seasonality that could improve the control of ASF

The European Commission requested EFSA to provide study designs for the investigation of four research domains according to major gaps in knowledge identified by EFSA in a report published in 2019: i) the patterns of seasonality of ASF in wild boar and domestic pigs in the EU; ii) the ASF epidemiology in wild boar; iii) ASF virus (ASFV) survival in the environment and iv) ASF transmission by vectors. In this Scientific Opinion, the first research domain on ASF seasonality is addressed. Therefore, five research objectives were proposed by the working group and broader ASF expert networks, such as ASF stop, ENETWILD, VectorNet, AHAW network and the AHAW Panel Experts. Of the five research objectives, only two were prioritised and elaborated into a general protocol/study design research proposal, namely: 1) to monitor the herd incidence of ASF outbreaks in EU Member States (MS) and 2) to investigate potential (seasonal) risk factors for ASF incursion in domestic pig herds of different herd types and/or size. To monitor the incidence in different pig herd types, it is advised to collect, besides ASF surveillance data, pig population data describing at least the following parameters per farm from the first moment of incursion in an affected MS: the numbers of pigs (e.g. number of breeding pigs sows and boars, weaners and fatteners) and the location and the type of farm (including details on the level of biosecurity implemented on the farm and the outdoor/indoor production). We suggest collecting data from all ASF‐affected MS through the SIGMA data model, which was developed for this purpose. To investigate potential risk factors for ASF incursion in domestic pig herds, we suggest a matched case–control design. Such a study design can be run either retrospectively or prospectively. The collected data on the pig herds and the ASF surveillance data in the SIGMA data model can be used to identify case and control farms.info:eu-repo/semantics/publishedVersio

First Detection of Salmonella enterica Serovar Choleraesuis in Free Ranging European Wild Boar in Sweden

Following the first detection of Salmonella enterica subsp. enterica, serovar Choleraesuis (S. Choleraesuis) in a Swedish pig herd for more than 40 years and subsequent detection of the same serotype in an enclosure with kept wild boar, a national surveillance for S. Choleraesuis in free living wild boar was launched. A total of 633 wild boar sampled within the active and the enhanced passive surveillance were examined for Salmonella enterica serovars by culture. Of these, 80 animals were culture positive for S. Choleraesuis var. Kunzendorf. All positive animals, including those in the original outbreaks, originated from counties located in the southern and eastern parts of Sweden. Fifty-eight isolates were selected for sequence typing, revealing a relatively homogenous population of S. Choleraesuis with two distinct genetic clusters containing isolates from the southern counties in one and the counties further northeast in the other. Sequenced isolates from domestic pig farms all clustered with wild boar in the same region. S. Choleraesuis appears highly contagious in dense wild boar populations, making it a relevant model for other infectious diseases that may be transmitted to pigs. The many potential routes of introduction and spread of S. Choleraesuis warrant further investigations in order to prepare for other disease threats

Evaluating the roles of directed breeding and gene flow in animal domestication

Peer reviewedPostprin

Mutations in the NS1 C-terminal tail do not enhance replication or virulence of the 2009 pandemic H1N1 influenza A virus

The ‘classical’ swine H1N1 influenza A virus lineage was established after the devastating 1918 human pandemic virus entered domestic pig herds. A descendent of this lineage recently re-emerged in humans as the 2009 pandemic H1N1 virus. Adaptation in pigs has led to several changes in the multifunctional viral NS1 protein as compared with the parental 1918 virus, most notably a K217E substitution that abolishes binding to host Crk/CrkL signalling adapters, and an 11 aa C-terminal truncation. Using reverse genetics, we reintroduced both these features into a prototype 2009 H1N1 strain, A/California/04/09. Restoration of Crk/CrkL binding or extension of NS1 to 230 aa had no impact on virus replication in human or swine cells. In addition, minimal effects on replication, pathogenicity and transmission were observed in mouse and ferret models. Our data suggest that the currently circulating 2009 H1N1 virus is optimized to replicate efficiently without requiring certain NS1 functions

Sigade Aafrika katku epidemioloogia Eestis ja ühe viirustüve iseloomustus

A Thesis for applying for the degree of Doctor of Philosophy in Veterinary Science.African swine fever (ASF) is an OIE-listed, dangerous viral disease of pigs, which has a devastating impact on animal health and the pig industry in many countries worldwide. During the period 2014–2020, ASF has been the main animal health issue in Estonia. This dissertation consists of three independent studies, which have been conducted with the aim to analyse the epidemiology of ASF and the course of the epidemic in the Estonian wild boar population, as well as in domestic pigs. In Study I, wild boar surveillance data (n = 7,015) collected from two distinct study areas from September 2014 to September 2016 were analysed. A statistically significant difference between the two areas in the temporal course of ASF seroprevalence was found. These findings indicate that ASF might have been introduced to the north-east of Estonia earlier then to the south of the country. The probability of detecting an ASF-positive animal was higher in young animals (< 1 year). Within wild boar found dead, there was a higher probability of finding an ASF-positive result compared to hunted animals. In Study II, the biological characteristics of the ASF virus strain (Est 14/WB) circulating in the wild boar population of north-east Estonia in 2014 were evaluated. Oronasal inoculation of ten young wild boar led to an acute and severe course of the disease in all infected animals. Nine animals died and one animal recovered completely from the disease. In conclusion, the ASFV strain was still found to be highly virulent. In Study III, the epidemiology of ASF in all 26 domestic pig outbreak herds that occurred in Estonia during the period 2015–2017 was retrospectively analysed. On most of the farms, the first clinical signs were mild and not ASF-specific despite the high virulence of the circulating virus. The highest mortality (29.7%) was seen on backyard farms (1–9 pigs) and the lowest (0.7%) on large commercial farms (> 1000 pigs). The spread of the virus within farms was slow and the contagiousness of the virus was relatively low. Farms of all sizes and types have been at risk. The results suggest that the increase in ASF cases in local wild boar populations is the main risk factor leading to the infection of farms; 88% of domestic outbreaks occurred in areas where ASF virus was detected in wild boar prior to the outbreak, within a radius of 15 km from the outbreak farm.Sigade Aafrika katk (SAK) on ohtlik sigade viirushaigus, mis põhjustab tõsiseid tagajärgi nii loomade tervisele kui ka majanduslikku kahju sektorile. Ajavahemikul 2014-2020 on SAK olnud peamine loomatervise probleem Eestis. Käesolev väitekiri koosneb kolmest eraldiseisvast uuringust, mis on viidud läbi eesmärgiga analüüsida SAK-i epidemioloogiat ning epideemia arengut Eesti metssigade populatsioonis ja kodusigadel. Esimeses uuringus analüüsiti kahest eraldiasuvast metssigade populatsioonist ajavahemikul septembrist 2014 kuni septembrini 2016 kogutud metssigade SAK andmeid (n=7015). Ilmnes, et SAK-i epidemioloogia oli neis kahes populatsioonis erinev. Seroloogiliste tulemuste ajaline analüüs näitas, et mediaan-aja mõju levimusele oli uuritavates piirkondades oluliselt erinev. Sellest tulenevalt võib eeldada, et SAK-i viirus ringles Kirde-Eestis enne, kui see Eestis ja piirkonnas ametlikult diagnoositi. Veel selgus, et tõenäosus leida SAK-positiivne metssiga oli kõrgem noorte loomade rühmas (vanus < 1 aasta) ning surnuna leitud loomade seas. Teises uuringus selgitati Kirde-Eestis 2014. aastal ringelnud SAK-i viirustüve (Est 14/WB) bioloogilisi omadusi. Katses nakatati oronasaalselt kümme umbes nelja kuu vanust metssiga. Kõigil loomadel tekkis äge haigestumine, neist üheksa looma surid ning üks tervenes haigusest täielikult. Katse tulemusel ei tuvastatud uuritud viirustüve virulentsuse langust. Kolmandas uuringus selgitati SAK-i epidemioloogiat kodusigadel. Selleks viidi epidemioloogiline uuring läbi kõigis (n = 26) seafarmides, kus ajavahemikul 2015–2017 diagnoositi SAK-i puhangud. Enamikus nakatunud karjades olid SAK-i esimesed kliinilised tunnused vaatamata haigustekitaja kõrgele virulentsusele leebed ning ebatüüpilised. Kõige kõrgemat suremust (29,7%) võis täheldada kodumajapidamistes (1-9 siga) ning kõige madalamat (0,7%) suurtes tootmisfarmides (>1000 siga). Viiruse levik nakatunud farmides oli aeglane ning nakkavus väike. Risk nakatuda tuvastati kõigis farmide suurusgruppides ning pidamisviiside juures. Uuringu tulemusel ilmnes, et SAKV-i levik kodusigade farmide ümbruskonna metssigade populatsioonis oli peamine riskitegur kodusigade nakatumiseks; 88% juhtudest leiti SAK-positiivseid metssigu puhangufarmist kuni 15 km kaugusel.The publication of this dissertation is supported by the Estonian University of Life Sciences

Population Densities and Disease Surveys of Wild Pigs in the Coast Ranges of Central and Northern California

In 1994 and 1995, 233 different wild pigs were captured during population research at seven research sites focused primarily in the coastal regions of central and northern California. Mark-resight data and information on wild pig movements were used to assess wild pig population densities at those sites. Population densities ranged from 1.01 wild pigs/km2 in Mendocino County in 1994 to 3.32 wild pigs/km2 in Santa Clara County in 1995. Comparisons of population densities between years at three research sites suggested that wild pig populations increased in 1995 in response to favorable forage conditions after the wet fall and winter of 1994-95. Serum samples collected from 462 wild pigs at 28 different sites were screened for exposure to brucellosis and pseudorabies. Preliminary results were that seropositive results for brucellosis were noted at only three sites, whereas no animals were confirmed seropositive for pseudorabies. Although analyses of these two diseases are continuing, test results for trichinellosis, toxoplasmosis, and sylvatic plague reinforce previous warnings to hunters and consumers that sanitary handling and cooking of wild swine meat are warranted

Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay

Das Afrikanische Schweinepest-Virus (ASPV) verursacht eine tödliche Viruserkrankung bei Schweinen. Dieses hat sich weltweit fortlaufend verbreitet und wurde im September 2020 erstmalig in Deutschland nachgewiesen. Der Ausbruch der Seuche kann schwere wirtschaftliche Verluste nach sich ziehen. Bis heute ist kein Impfstoff zugelassen, daher sind Überwachung der epidemiologischen Situation und der frühzeitige Erregernachweis unerlässlich für die Bekämpfung der Afrikanischen Schweinepest als Tierseuche. Die Polymerase-Kettenreaktion (PCR) gilt als Goldstandard für den Nachweis von ASPV und zeichnet sich durch eine hohe Sensitivität und Spezifität aus. Allerdings erfordert die PCR gut ausgestattete Testlabore und ist zeitintensiv. Point-of-Need-Tests können schnelle und zuverlässige direkt vor Ort liefern und stellen somit eine Alternative zum Goldstandard PCR dar. Ziel dieser Studie war es, einen Point-of-Need-Test zum Nachweis von ASPV zu entwickeln. Dieser beruht auf der Grundlage der Rekombinase-Polymerase-Amplifikation (RPA) und sollte vor Ort einsatzfähig sein. Es wurden drei Primersätze und eine Sonde auf der Grundlage des B646L-Gens, welches für das virale Kapsidprotein p72 vom ASP-Virus kodiert, entwickelt. Alle möglichen Kombinationen wurden getestet. Die analytische Sensitivität wurde mit acht Wiederholungen von Verdünnungsreihen des molekularen Standards (102-100 DNA-Kopien pro µl) ermittelt. Die Nachweisgrenze wurde anhand einer Probit-Analyse dieser Durchläufe berechnet. Die Spezifität wurde mit verschiedenen viralen Nukleinsäuren von anderen das Schwein infizierenden Erregern überprüft. Um den Test im Feld einsatzfähig zu gestalten, wurden mittels ASPV-RPA zwei verschiedene Extraktionsansätze mit allen 73 verfügbaren Schweineblutproben getestet: eine schnelle Hitze/Lysepuffer-Extraktionsmethode und ein standardisiertes Extraktionsverfahren auf Spin-Säule-Basis. Die diagnostische Sensitivität und Spezifität wurde für beide Testverfahren berechnet. Alle Ergebnisse wurden mit einer etablierten real-time PCR für ASPV verglichen. Eine kleine Pilotstudie zum Feldeinsatz des ASPV-RPA-Tests wurde in Uganda mit 20 Blutproben unter Verwendung des Kofferlabors durchgeführt. Die berechnete Nachweisgrenze von ASPV-RPA lag bei 3,5 DNA-Kopien pro µl. Alle untersuchten ASPV-Genotypen wurden detektiert, aber keine anderen viralen Nukleinsäuren. Bei Verwendung der standardisierten DNA-Extraktionsmethode mit anschließender Durchführung der ASPV-RPA lag die diagnostische Sensitivität und Spezifität bei 100%, wie auch mittels der real-time PCR. Auch das schnelle Hitze-/Lysepuffer Protokoll zeigte vielversprechende Ergebnisse und erreichte eine Positivrate von 97% mittels ASPV-RPA im Vergleich zu 38% bei der PCR. In Uganda wurden elf ASPV-RPA-Proben als positiv erkannt, darunter zwei fieberfreie asymptomatische Tiere. Der schnelle Erregernachweis stellt einen essenziellen Aspekt der ASP Seuchenbekämpfung dar. Die ASPV-RPA erwies sich als genauso empfindlich und spezifisch wie die Goldstandard-PCR zur Erregeridentifizierung. In Kombination mit dem Schritt der DNA-Extraktion durch Hitze/Lysepuffer benötigt der entwickelte Test etwa 25 Minuten von der Probenentnahme bis zum Ergebnis. Die Positivrate ist mit 97% vielversprechend, wobei die ASPV-RPA im Vergleich zur PCR eine höhere Toleranz gegenüber Inhibitoren aufwies. Wie die Pilot-Feldstudie in Uganda mit dem Kofferlabor zeigt, ist ASPV-RPA eine im Feld einsatzfähige Nachweismethode. Das Kofferlabor bedarf lediglich einer Grundausstattung und einer Solarbatterie. Somit stellt das Kofferlabor eine vielversprechende Diagnostikmethode dar, welche vor Ort in ressourcenarmen Umgebungen zum Nachweis des ASPV eingesetzt werden kann.:1. Introduction 2. Literature overview 2.1 African swine fever 2.1.1 Aetiology 2.1.1.1 Classification and taxonomy 2.1.1.2 Viral structure and genome 2.1.1.3 Genetic typing and antigenic variability 2.1.2 Epidemiology 2.1.2.1 Disease distribution 2.1.2.2 Host range and epidemiological cycles 2.1.2.2.1 Warthog-tick cycle 2.1.2.2.2 Domestic pig-tick cycle 2.1.2.2.3 Domestic pig cycle 2.1.2.2.4 Wild boar-environment cycle 2.1.2.3 Tenacity, transmission, and infectivity 2.1.3 Pathophysiology 2.1.3.1 Pathogenesis 2.1.3.2 Clinical signs and pathological findings 2.1.3.3 Differential diagnosis 2.2 Available diagnostic tools for ASFV 2.1.4 Diagnosis based on immune response 2.1.5 Diagnosis based on agent identification 2.3 Gaps in African swine fever diagnostics 3. Publication 3.1 Statement of contribution 3.1.1 Publication 4. Discussion 5. Summary 6. Zusammenfassung 7. References 8. Appendix 9. AcknowledgementsAfrican swine fever virus (ASFV) causes a deadly viral disease in pigs. The virus has gradually spread throughout the world and was reported in Germany in September 2020. ASF outbreak can lead to huge economical loss. No vaccine is commercially available and thus, surveillance and early detection play a pivotal role to control an ASF outbreak. Polymerase Chain Reaction (PCR) is considered the gold standard for ASFV detection due to its superior sensitivity and specificity. However, it is time-consuming and requires well-equipped laboratories. Point-of-need tests can offer an alternative, delivering fast and reliable results directly in the field. The aim of this study was to establish a field-deployable point-of-need test based on Recombinase Polymerase Amplification (RPA) to detect ASFV. Material and Methods: Three sets of primers and one probe based on the B646L gene which encodes for the viral capsid protein p72 were designed. All possible combinations were screened. Analytical sensitivity was tested with eight replicates of serial dilutions of the molecular standard (102-10° DNA copies per µl). The limit of detection was calculated using probit analysis. ASFV-RPA’s specificity was tested using various viral nucleic acids of pathogens infecting pigs. To allow the deployment at point of need, two different extraction approaches were tested in ASFV-RPA with all 73 pig blood samples included in this study: a rapid heat/lysis buffer extraction method and a standardized spin-column based extraction kit. Diagnostic sensitivity and specificity were calculated for both test approaches. All results were compared to an established real-time PCR for ASFV. A small pilot study for ASFV-RPA assay deployment was done in Uganda with 20 blood samples of a suspected outbreak using the field-deployable suitcaselab. The calculated limit of detection of ASFV-RPA was 3.5 DNA copies per µl. All screened ASFV genotypes were detected while no other viral nucleic acids were identified. Using the standardized DNA extraction method in ASFV-RPA, and compared to real-time PCR, diagnostic sensitivity and specificity were 100%. The rapid heat/lysis buffer protocol showed very promising results, achieving 97% of positivity rate compared to a 38% of the real-time PCR. In Uganda, ASFV-RPA detected 11 samples as positive, including two known afebrile animals. Immediate agent detection is a key aspect of ASF outbreak control. ASFV-RPA is as sensitive and specific as a gold standard PCR for ASFV identification. Combined with the heat/lysis buffer DNA isolation step, the duration of the assay is around 25 minutes from sample collection to result readout, with a promising positivity rate of 97% which indicates tolerance against inhibitors. ASFV-RPA is a portable detection method, as revealed during the pilot field study in Uganda. Only requiring basic equipment and solar batteries, the suitcase lab is a promising tool for on-site diagnostics in resource limited settings to detect ASFV.:1. Introduction 2. Literature overview 2.1 African swine fever 2.1.1 Aetiology 2.1.1.1 Classification and taxonomy 2.1.1.2 Viral structure and genome 2.1.1.3 Genetic typing and antigenic variability 2.1.2 Epidemiology 2.1.2.1 Disease distribution 2.1.2.2 Host range and epidemiological cycles 2.1.2.2.1 Warthog-tick cycle 2.1.2.2.2 Domestic pig-tick cycle 2.1.2.2.3 Domestic pig cycle 2.1.2.2.4 Wild boar-environment cycle 2.1.2.3 Tenacity, transmission, and infectivity 2.1.3 Pathophysiology 2.1.3.1 Pathogenesis 2.1.3.2 Clinical signs and pathological findings 2.1.3.3 Differential diagnosis 2.2 Available diagnostic tools for ASFV 2.1.4 Diagnosis based on immune response 2.1.5 Diagnosis based on agent identification 2.3 Gaps in African swine fever diagnostics 3. Publication 3.1 Statement of contribution 3.1.1 Publication 4. Discussion 5. Summary 6. Zusammenfassung 7. References 8. Appendix 9. Acknowledgement

Trichinelosis in Animals

Trichinellosis (also trichinosis) in animals is caused by nematodes (roundworms) of the family Trichinellidae Ward, 1970. Family characteristics. Parasites with small bud right body. They have not sexual pterygas. The anus is opened at the terminal part of the body. The cloaks are opened at the ¼ frontal part of the body. The adult females are larva-productive. They are parasites of the intestinal system. Trichinella spiralis (Trichina spiral) Owen, 1833. Eight species of Trichinella are now recognized, based on host (Kapel, C M O 2000; Krivokapich, S J; Pozio E and D S Zarlenga, 2005; Pozio E et al, 1992), but the most important for animals domestic are: T. spiralis found as parasitic diseases in humans, pigs, rodents, and many carnivorous animals, of Europe, Asia, North America, with specific pathologies in pigs. T. native parasite of wild carnivorous of Euro – Asiatic areas northern of parallel 40°. It is specific diseases of carnivorous and omnivorous animals. T.nelsoni found as a parasite of wilds carnivorous animals of Asiatic areas southern of parallel 40°. T. pseudospiralis is parasite of cats, rodents, and pigs. It is recognized from other species because of the adult forms have smaller dimensions and forms noncapsulated cists. T. spiralis is the cause of Trichinellosis, one of most important zoonosis all over the world. It is found worldwide in many carnivorous and omnivorous animals, insectivorous animals, rodents, wilds animals and humans (Pozio E and G Marucci 2003). It was found at 103 mammals. Occasionally may be found as a parasite of horses. Developmental traits of T. spiralis is that infested hosts initially are final hosts because of they host adults forms at their intestine, but later on, they are presented as an intermediate host, because of they host larval forms at their muscles. Today’s identification of samples to the species level and genotyping are based primarily upon molecular means (Pozio, E., and G. Marucci. 2003).Keywords: trichinosis, Trichinella larvae, tropism, trichinelloscopic examination, trypsine techniques, xenodiagnostic experiments, etc